An alcohol-sensing site in the calcium- and voltage-gated,large conductance potassium (BK) channel

Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol–BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel’s physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.Alcohol (ethyl alcohol, ethanol) is a psychoactive agent that has been overwhelmingly consumed by mankind across cultures and civilizations. Alcohol actions on central nervous system (CNS) physiology and behavior are largely independent of beverage type but due to ethanol itself (1). Ethanol alters cell excitability by modifying function of transmembrane (TM) ion channel proteins, including K⁺ channels. These channels constitute the most heterogeneous and extensive group of ion channels, its members belonging to TM2, TM4, and TM6 protein superfamilies. Within this myriad of proteins, several K⁺ channels have been shown to modify behavior in response to acute exposure to ethanol concentrations that reach the CNS and other excitable tissues during alcohol drinking (2–5). However, with the sole exception of the TM2, G protein-regulated inward rectifier K⁺ (GIRK) channel (6), there is no structural information on ethanol-K⁺ channel protein interacting sites currently available.Voltage/Ca²⁺-gated, large conductance K⁺ channels (BK), which are members of the TM6 voltage-gated ion channel superfamily, constitute major mediators of alcohol actions in excitable tissues. Acute exposure to ethanol levels reached in CNS during alcohol intoxication alters BK-mediated currents and thus, elicits widespread and profound modifications in physiology and behavior. In rodent models, acute ethanol exposure leads to reduced vasopressin, oxytocin and growth hormone release with consequent perturbation in physiology and behavior (7), altered firing rates in nucleus accumbens (8) and dorsal root ganglia neurons (9), and alcohol-induced cerebral artery constriction (10, 11). Moreover, studies in both mammals and invertebrate models demonstrate that ethanol targeting of neuronal BK is involved in development of alcohol tolerance and dependence (12–16). Although the physiological and behavioral consequences of ethanol disruption of BK function have been well documented, it remains unknown whether alcohol modification of BK function results from drug interaction with a defined recognition site(s) in a protein target vs. physical perturbation of the proteolipid environment where the BK protein resides. Thus, location and structural characteristics of the ethanol-recognition site(s), as well as nature of chemical bonds between ethanol and functional target that lead to modification of BK function, remain unknown.Ethanol-induced regulation of BK channels is fine-tuned by many factors, including the BK channel-forming slo1 protein (α subunit) isoform (17) and its modification by phosphorylation (18), BK channel accessory (β) subunits (11), the channel-activating ionic ligand (Cai2+) (19) and the lipid microenvironment around the BK protein complex (20). However, ethanol perturbation of BK function is sustained when the slo1 protein is probed with the alcohol in cell-free membrane patches (19–21) or after protein reconstitution into artificial lipid bilayers (22). We recently demonstrated that perturbation of slo1 function by ethanol concentrations reached in blood during alcohol intoxication does not extend to Na⁺-gated slo2 and pH-gated slo3 channels, which are phylogenetically and structurally related to slo1. However, ethanol sensitivity does extend to a prokaryotic K⁺ channel from Methanobacterium thermoautotrophicum (MthK) (23), a TM2 ion channel that shares basic Cai2+-driven gating mechanisms with slo1 (24). Collectively, these studies lead us to hypothesize that ethanol-recognition site(s) involved in alcohol modification of BK current exists in the slo1 cytosolic Cai2+-sensing tail domain (CTD).Based on crystallographic data of the slo1 CTD and primary alignment of slo1-related ion channels that share ethanol sensitivity, we first identified eight putative ethanol recognition regions in the slo1 CTD. Using computational modeling, point amino acid substitutions and electrophysiology, we identified a distinct pocket as the ethanol-recognition site that leads to alcohol modification of BK current. This site has a few common characteristics of alcohol-binding protein sequences (25), yet presents features that differ from those of the alcohol site described in GIRK (6). In opposition to GIRK currents, which can be potentiated by alcohol in absence of G proteins (6), ethanol modulation of BK currents is dependent on the presence of Cai2+ (19). Our data strongly suggest that ethanol access to the newly identified BK ethanol-recognition site depends on the Cai2+ levels associated with the slo1 CTD. Thus, current data not only provide a structural basis for understanding Cai2+–alcohol allosterism on BK channels but could render structural insights on other ligand-gated channels that are activated by ethanol in presence of their natural ligand (26–30). Finally, present data document that the newly identified site plays a critical role in BK channel sensitivity to long-chain alkanols and explain the reported chain length differential sensitivity (“cutoff”) of linear n-alkanols to modify BK current (31).Identification of a distinct alcohol-sensing site in BK channels opens the door for rational design of pharmaceuticals to counteract widespread effects of alcohol intoxication in the body without altering basal BK channel function. Because this site is present in human BK protein ({"type":"entrez-protein","attrs":{"text":"AAA92290.1","term_id":"758791","term_text":"AAA92290.1"}}AAA92290.1), it is possible that genetic, epigenetic or other modifications of the alcohol-sensing site in BK channels could contribute to differential sensitivity to alcohol intoxication in humans. In addition, considering that individuals with low alcohol sensitivity are prone to developing heavy drinking (32), an altered profile of alcohol-sensing site on BK channels might be included as a potential predictor, along with other targets, for developing alcohol preference.     

EFFICACY OF RAMIPRIL VERSUS ENALAPRIL IN PATIENTS WITH MILD TO MODERATE ESSENTIAL HYPERTENSION

SUMMARY This double-blind, randomised, cross-over study investigated the antihypertensive efficacy of ramipril and enalapril was completed by 30 patients with mild-to-moderate essential hypertension. After a four-week placebo run-in phase, the patients received either 2.5mg ramipril or 10mg enalapril once daily for four weeks. The dosages were increased to 5mg ramipril and 20mg enalapril for a further four weeks. After a placebo washout phase of four weeks, the patients were crossed over to the alternative treatment. The decrease in average 24-hour ambulatory diastolic blood pressure from week 0 to week 8 was 1.6mmHg greater with ramipril than enalapril (90% confidence interval 0.6-2.7mmHg). The corresponding reduction in for systolic blood pressure was also greater with ramipril than enalapril by 2.4mmHg (90% confidence interval: 0.5-4.2mmHg). For the difference in the drop of 24-hour ambulatory diastolic blood pressure between ramipril and enalapril the lower level of the 90% confidence interval (CI) is above the clinically relevant difference of -3mmHg. This is an indication that ramipril (2.5 and 5mg dose) is at least as effective as enalapril (10 and 20mg dose) in decreasing blood pressure in patients with mild-to-moderate essential hypertension. The duration of adequate antihypertensive effect was relatively long for both ramipril and enalapril; however, ramipril tended to have a more prolonged antihypertensive effect. Ramipril had a higher diastolic and systolic trough/peak ratio than enalapril, resulting in a more uniform antihypertensive effect over the 24-hour treatment period. Both ramipril and enalapril were well tolerated and the two treatment groups had similar safety profiles.     

Alloimmunization to platelet-specific antigens on glycoproteins IIb- IIIa and Ib/IX in multiply transfused thrombocytopenic patients

The rate of alloimmunization to platelet-specific antigens associated with platelet glycoproteins (GPs) IIb-IIIa and Ib/IX was studied in 293 multiply transfused thrombocytopenic patients. Antibodies to platelet-specific antigens were measured with a solid-phase assay using platelet GP IIb-IIIa or Ib/IX as the antigenic targets. Nine patients were found to have antibodies to platelet GP IIb-IIIa, and no patients had antibodies to platelet GP Ib/IX. In six of these nine patients, the specificity of the antibody was shown by using GP IIb-IIIa from donors with different platelet-specific antigen phenotypes. In the remaining three patients with antibodies to platelet GP IIb-IIIa, no specificity could be identified. These patients had autoimmune thrombocytopenia in association with lymphoma. The alloimmunization rate to platelet-specific antigens associated with GP IIb-IIIa was 2 percent, whereas the rate of alloimmunization to HLA antigens was 23 percent. Of the patients alloimmunized to HLA antigens, 9 percent also had antibodies to platelet-specific antigens. A poor response to HLA-identical platelet transfusions was observed only in those patients with positive assays in the solid-phase test. These results suggest that the incidence of antibodies to platelet-specific antigens carried on GP IIb-IIIa is low. Platelet-specific antibodies may be found more frequently in patients alloimmunized to HLA antigens than in those not so alloimmunized.     

Effect of white cells on platelets during storage

White cells (WBCs) present in stored platelet concentrates have adverse effects on platelet function and on posttransfusion recovery. Although these effects have been attributed to the fall in pH that results from active WBC metabolism, platelets stored in gas-permeable storage bags still exhibit abnormalities, despite maintenance of a stable pH of greater than 6.0. The changes in platelet proteins and function brought about by storage with a controlled number of WBCs were studied. Twelve platelet-pheresis specimens were centrifuged at 180 x g to achieve a WBC count of less than 2 x 10(5) per mL (which contained less than 10% granulocytes). These specimens were split into two aliquots and placed in platelet bags for storage at 22 degrees C with constant horizontal agitation. Neutrophils, obtained from the same donor by centrifugation of 50 mL of whole blood through a discontinuous ficoll gradient, were added to one of the two platelet storage bags to achieve a final neutrophil count of 1 x 10(6) per mL. Platelet aliquots were removed and studied on Days 3 and 5. In platelets stored without neutrophils, the average response to ristocetin, using the mean slope as an index of platelet responsiveness, was 10.3 (n = 9, SD = 11) on Day 3, whereas for the platelets stored with neutrophils, it was 1.25 (n = 12, SD = 0.9, p less than 0.01). Significant differences were also seen on Day 5 (slope = 4.5 for platelets stored without neutrophils, slope = 0.3 for platelets stored with neutrophils, p less than 0.01). Platelet aggregation with 8 microM ADP and 1.5 mg per mL of collagen did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

白介素与溃疡性结肠炎

近年来对白介素(interleukin,IL)和溃疡性结肠炎(ulcerative colitis,UC)的研究取得了很大进展,我们通过总结整理以前有关IL和UC的文献,概括出IL的产生和在UC发病及病理变化中的作用机制:IL-1直接介导了UC初期阶段炎症的发生:IL-8、IL-6直接促进炎性细胞过度分泌和/或抑制了炎性细胞的凋亡,IL-2分泌减少导致免疫系统内细胞间网络调节失衡, 使局部炎症介质和自由基释放,引起细胞毒作用,IL主要通过影响机体整体和/或局部免疫系统的功能介导UC的产生,并与UC的迁延难愈和反复发作有关．